Twin-Engine Janus Supramolecular Nanomotors with Counterbalanced Motion

Supramolecular nanomotors were created with two types of propelling forces that were able to counterbalance each other. The particles were based on bowl-shaped polymer vesicles, or stomatocytes, assembled from the amphiphilic block copolymer poly(ethylene glycol)-block-polystyrene. The first method of propulsion was installed by loading the nanocavity of the stomatocytes with the enzyme catalase, which enabled the decomposition of hydrogen peroxide into water and oxygen, leading to a chemically induced motion. The second method of propulsion was attained by applying a hemispherical gold coating on the stomatocytes, on the opposite side of the opening, making the particles susceptible to near-infrared laser light. By exposing these Janus-type twin engine nanomotors to both hydrogen peroxide (H2O2) and near-infrared light, two competing driving forces were synchronously generated, resulting in a counterbalanced, “seesaw effect” motion. By precisely manipulating the incident laser power and concentration of H2O2, the supramolecular nanomotors could be halted in a standby mode. Furthermore, the fact that these Janus stomatocytes were equipped with opposing motile forces also provided a proof of the direction of motion of the enzyme-activated stomatocytes. Finally, the modulation of the “seesaw effect”, by tuning the net outcome of the two coexisting driving forces, was used to attain switchable control of the motile behavior of the twin-engine nanomotors. Supramolecular nanomotors that can be steered by two orthogonal propulsion mechanisms hold considerable potential for being used in complex tasks, including active transportation and environmental remediation.

N anoscopic particles with motile features have become a topic of intensive investigation over the past years. Inspired by natural motor systems, a range of different particles with a variety of propulsion mechanisms have been developed. 1−5 They can convert local energy from their surroundings into locomotion in a fluidic environment, which can be utilized for the completion of complex tasks. 6−10 Customized autonomous nanomotors have been created for sensing, 11,12 environmental remediation, 13,14 energy (hydrogen−oxygen fuel cell), 15 and biomedical applications. 16−20 The majority of first-generation nanomotors was propelled by catalytic decomposition of chemical fuels, such as the bubblepair propelled colloidal kayakers. 21 However, in fuel-deprived environments, the motion of this kind of nanomotor is inevitably suppressed. To tackle this issue, fuel-free motors that can be powered and remotely guided by external physical stimuli (e.g., magnetic fields, ultrasound, and light) have been successfully constructed. 22−25 Most of the current artificial nanomotors are however still powered by a single engine, which is accompanied by some limitations.
A major shortcoming of a single-mode nanomotor is the difficulty in precisely manipulating and modulating the motile behavior on-demand. 26,27 To address this challenge, a new generation of nanomotors with multimode propulsion thus needs to be further developed. Considerable efforts have been devoted to realizing this class of artificial motors. 28,29 To date, several dual-driven micro/nanomotors based on hybrid materials have been created, which are propelled by chemical−ultrasound, 30 chemical−magnetic, 31 chemical− light, 32,33 ultrasound−magnetic, 34 and ultrasound−light energy sources. 35 Compared to the single-engine micro/nanomotors, incorporation of two propulsion mechanisms in one motor makes them more robust in complex surroundings and less affected by the constraints on the availability of fuel resources or other environmental parameters. Importantly, besides addressing the limitations of traditional single-mode motors, two propulsion modes working together enable a better control over the directionality and precision of motion, thereby expanding the application potential of micro/nanomotor systems. However, among these dual-driven micro/nanomotors, only a few were reported to have a controllable stalling of motion through the "seesaw effect", where opposing motile forces counterbalance each other.
To allow directed motion, asymmetry should be installed in the particle design. A very effective approach is the construction of Janus polymeric particles/capsules with a hemispherical platinum or gold shell; upon exposure to hydrogen peroxide (H 2 O 2 ) or near-infrared (NIR) laser irradiation, respectively, autonomous motion is introduced. 36−39 Another useful chassis for nanomotor design was recently developed by our group and is based on stomatocytes. Stomatocytes are polymeric vesicles with a unique bowl-shaped morphology, created via an osmotic-induced shape change process of spherical vesicles composed of amphiphilic block copolymers. 40−43 By loading the well-defined cavity with catalytic nanoparticles or enzymes, stomatocyte-based catalytic nanomotors were created. 44,45 Even enzymatic networks could be effectively incorporated for this purpose. 46 The introduction of Janus morphology on an enzyme-filled stomatocyte would allow the construction of an efficient dual-powered supramolecular nanomotor.
Herein, we present a twin-engine supramolecular nanomotor, which is based on stomatocytes assembled from the amphiphilic block copolymer poly(ethylene glycol)-blockpolystyrene (PEG-b-PS). The responsiveness to chemical fuel was included by loading the stomatocyte nanocavity with the enzyme catalase during the process of shape transformation. For the installment of a second driving force, a hemispherical gold layer was introduced onto the stomatocytes by sputter coating, on the other side of the opening, which made the particles susceptible to NIR light. The dual-functional nanomotors displayed efficient propulsion upon either irradiation with NIR light (NIR driven mode) or in the presence of H 2 O 2 (enzyme driven mode). A "seesaw effect" was observed when both forces were applied simultaneously, since the NIR driving force generated on the gold side counterbalanced the enzymatic propulsion force. Because of the observed "seesaw effect", we experimentally confirmed the movement direction of the enzyme-propelled stomatocytes with the cavity pointing away from the direction of motion. More importantly, a high level of control over motion was achieved by tuning the net outcome of the two coexisting driving forces via regulating the incident laser power.
To investigate the twin-engine nanomotor features, three different supramolecular nanomotors were constructed: stomatocytes coated with a hemispherical gold layer (motor 1: Janus stomatocytes), which could be propelled solely by NIR light; catalase-filled stomatocytes (motor 2: catalase stomatocytes), which responded to H 2 O 2 , and the combined system in which both functionalities were included. Their method of preparation is schematically depicted in Figure 1. All stomatocytes were assembled from the amphiphilic block copolymer poly(ethylene glycol)-polystyrene (PEG 45 -b-PS 230 ), which was synthesized by using atom-transfer radical polymerization as reported in previously published work ( Figure S1). 47 The formation of the bowl-shaped stomatocytes was conducted according to established protocols. In short, after assembling the block copolymer into spherical vesicles, by addition of water to a polymer solution in organic solvent, dialysis was conducted to initiate an osmotic shock-induced shape change process. 43 The morphology of the stomatocytes was confirmed by scanning electron microscopy (SEM), as shown in Figure S1C. To prepare the Janus stomatocytes, the above-mentioned method was modified. Before the shape change process was employed, the spherical polymersomes were deposited on a silica wafer into a monolayer via dropcasting. Subsequently, this was followed by sputter coating of a gold (Au) layer on top of the polymersomes. The Janus polymersomes were released from the substrate into solution via ultrasound treatment. Next, they were transferred to a dialysis bag to induce the shape transformation ( Figure 2A).  Due to the presence of the hemispherical gold layer, only the uncoated polymer side showed sufficient flexibility to undergo a shape transformation, which resulted in indention of the vesicles mainly on the opposite side of the gold layer. This specific morphology was observed by SEM and transmission electron microscopy (TEM) (Figures 2B−D and S2). Energydispersive X-ray (EDX) mapping of Au on a number of Janus stomatocytes further confirmed the existence of a hemispherical Au layer on the "bowl bottom" part of the stomatocyte, away from the opening ( Figure 2E,F). Dynamic light scattering (DLS) data furthermore indicated that the average hydrodynamic size of Janus stomatocytes was almost comparable to those of native stomatocytes, 435.3 and 410.7 nm with polydispersity index of 0.107 and 0.027, respectively ( Figure S3).
By coating stomatocytes with a hemispherical Au coating, Janus stomatocytes were obtained, which were photoactivatable via the surface plasmon resonance features of the Au layer. According to established theories, thermophoresis is thereby the main propulsion mechanism. 48−51 Under NIR irradiation, the temperature of the surrounding medium around a Janus particle is spatially nonuniform, resulting in inhomogeneous thermal fluctuations, which leads to particle motion. To investigate the movement behavior, two photon-confocal laser scanning microscopy (TP-CLSM) was used to observe and record the laser power-dependent motion. The trajectory of randomly selected Janus stomatocytes (the number of nanomotors n = 20) was tracked from the recorded video by ImageJ ( Figure S4). The motion of Janus stomatocytes was indeed laser power dependent, since with the increase of output laser power, a higher velocity was achieved ( Figure  2G). The mean squared displacement (MSD) as a function of NIR laser power was calculated according to previously published methods, 52,53 which further confirmed that the motion of Janus stomatocytes is strongly reliant on the incident laser power ( Figure 2H).
As a second control, enzyme-filled stomatocytes (motor 2: catalase stomatocytes) were prepared according to earlier published protocols. Catalase was used, as it is highly efficient in decomposing H 2 O 2 into water and oxygen, and has often shown its value in the research of active particles. 54,55 As catalase is an enzyme very sensitive to organic solvents, we adopted a mild methodology, developed in our group, to load catalase into the nanocavity of stomatocytes. 44,45 Briefly, spherical polymersomes were prepared by the solvent exchange method, followed by dialysis-induced shape transformation to stomatocytes. This process was quenched in an early stage by the addition of an excess of water to attain stomatocytes with a wide-open neck. Then, the shape change process was continued in the presence of catalase and a small fraction of organic solvent to resolubilize the membrane. After the shape change process, the opening of the stomatocytes was significantly diminished to prevent enzyme leakage. Morphological and size changes were followed by SEM and DLS, respectively ( Figure S5). Asymmetric flow field flow fractionation (AF4) coupled to multiangle light scattering and DLS were used to confirm the encapsulation of the enzyme ( Figure S6). The motility of the catalase stomatocytes as a function of H 2 O 2 concentration was determined by TP-CLSM. Figure S7A displays tracks of randomly selected enzyme nanomotors in the presence of H 2 O 2 . Based on the trajectories, an average velocity and MSD of catalase stomatocytes were calculated (Figures S7B and S7C). These data demonstrate that the motion of catalase stomatocytes was H 2 O 2 concentration dependent, since more directional motion and higher speeds were achieved when increasing the H 2 O 2 concentration.
Next, the two propulsion mechanisms were combined in the same particle, yielding twin-engine Janus supramolecular nanomotors (motor 3: catalase/Janus stomatocytes, Figure  3A). We first created Au-coated Janus polymersomes as described for the preparation of motor 1; these were shape changed into wide-necked Janus stomatocytes. Then, the same protocol for entrapment of catalase into stomatocytes was conducted, as described for motor 2. SEM measurements confirmed the overall shape transformation from spherical Janus polymersomes and wide-open Janus stomatocytes to narrow neck Janus stomatocytes ( Figures 3B,C and S8). To analyze the efficiency of catalase encapsulation, we compared the enzyme loading and activity between stomatocytes and Janus stomatocytes using a BCA protein assay and catalase activity assay (Figures S9 and S10). From these data, we experimentally verified that both types of stomatocytes showed similar enzyme features. The size of the Janus stomatocytes was slightly increased during the process possibly because of the presence of the hemispherical Au coating.
First, the propulsion performance of the catalase/Janus stomatocytes under single-mode conditions was investigated using TP-CLSM ( Figures 3D,E and S11). To enhance the traceability of the catalase/Janus stomatocytes, doxorubicin (Dox) was used as a fluorescent dye, which was loaded during the formation of polymersomes. Under NIR irradiation, catalase/Janus stomatocytes were photoactivated and exhibited the expected behavior, namely, an increase in velocity, MSD, and moving distance with enhanced laser power. Next, we investigated the enzyme-activated motion by the addition of H 2 O 2 fuel at different concentrations (0.15 wt % H 2 O 2 and 0.75 wt % H 2 O 2 ). The directed motion of the enzyme nanomotors was strongly dependent on the fuel concentration. Upon increasing the concentration of H 2 O 2 , particle velocity and MSD were enhanced.
Next, the nanomotors were exposed to both driving forces. To simplify the analysis process, we investigated the movement behavior of catalase/Janus stomatocytes as a function of NIR laser power in the presence of a fixed concentration of H 2 O 2 fuel. When the experiment was performed in the presence of 0.15 wt % H 2 O 2 , particle velocity increased with the increasing NIR output laser power. However, when the same experiment was performed at 0.75 wt % H 2 O 2 , the velocity first decreased, before an increase could be observed at a higher NIR laser power ( Figure 3D,E), leading to a minimum particle velocity at a specific fuel concentration and NIR laser power. This behavior is explained by the fact that the driving forces generated by catalytic decomposition of H 2 O 2 and the NIRinduced photothermal effect oppose each other, resulting in a "seesaw effect" of the supramolecular nanomotors ( Figure  S12A). As both the enzyme-driven system and the photothermal effect have to be directional to counterbalance each other, this experiment also provides direct proof of the motion direction of enzyme-propelled stomatocytes. The photothermal effect creates a temperature gradient around the gold shell, which drives the particles away from the source of heating; the particles move with the stomatocyte cavity to the front and the Au layer to the back. This motion can only be compensated if the enzyme-driven propulsion does exactly the opposite. This means that the cavity is pointing to the rear, from which oxygen or oxygen bubbles can escape to install directed motion in the stomatocytes.
Having established the conditions under which the two forces were in balance, we could create a "stop-and-go" situation by switching the laser power on and off, respectively (Figures 4 and S12). This method of control was easier to achieve than to tune the H 2 O 2 fuel concentration.
Based on the studies reported in Figure 3, the nanomotors were exposed to three different output laser powers, 0, 30.1, and 35.7 mW, as the latter two should allow minimal motion when applied in the presence of 0.75 wt % H 2 O 2 . As shown in Figure 4A, catalase/Janus stomatocytes displayed regular fuel concentration-dependent motion behavior in the absence of NIR laser irradiation. The average speed of catalase/Janus stomatocytes was increased from 1.31 to 3.09 μm/s with the increasing H 2 O 2 concentration (Supporting Information, Videos S1 and S2). An apparent decrease in velocity was observed after switching the NIR laser on ( Figure 4B,C). Movement trajectories of catalase/Janus stomatocytes extracted from recorded videos (Supporting Information, Videos S3−S5) displayed that the nanomotors indeed entered a "static state" by exposing them both to 0.75 wt % H 2 O 2 fuel and NIR laser light of certain laser power (30.1 and 35.7 mW). Inset figures in Figure 4 depict the mechanism of the "seesaw effect" induced by opposing forces. This method provides excellent control over the motion of the particles.
To further demonstrate the ability to control stomatocytes' motion using light and chemical fuel, nanoparticle tracking analysis equipped with an external laser source was utilized, as shown in Figure 5A. Here, we aimed at studying the ability of the stomatocytes to be activated and deactivated using light in the presence of H 2 O 2 . Indeed, MSD analysis and motion trajectories of the nanomotors showed alteration of motion between non-Brownian and Brownian upon switching the light on and off, respectively. Three different driven modes existed; these included two single modes (i.e., laser-and enzyme-driven modes), as well as a dual mode (i.e., two driving forces coexisting in one nanomotor). In the absence of H 2 O 2 , the catalase/Janus stomatocytes were propelled in single mode (laser-driven mode), resulting in laser power-dependent motion ( Figure 5B, Supporting Information, Video S6). In the presence of H 2 O 2 and absence of light, the motion of catalase/Janus stomatocytes exhibited the enzyme-driven mode, as presented in Figure 5C. Precise motion control was achieved by switching between single and dual driven modes, as demonstrated by velocity, MSD, and motion trajectories of the nanomotors (Figures 5D and S13, Supporting Information, Video S7). It is worth mentioning that the directional motion of such a stomatocyte platform is in line with previous reports. 56 Additionally, the importance of the gold layer for light-mediated motion was confirmed by investigating the behavior of uncoated stomatocytes (without gold) upon laser irradiation ( Figure S14). MSD and trajectory analysis did not show any difference in motion of such uncoated stomatocytes upon light irradiation.
Finally, we investigated the collective behavior of the catalase/Janus stomatocytes. Pioneering work demonstrated that self-propelled particles prefer to accumulate in a region of space and have a strong tendency to form clusters, compared to passive particles. 57−62 For instance, light-propelled micromotors based on the photothermal effect formed cluster structures, due to light-induced convection. 63−65 Convection is generated in the liquid surrounding the particles, due to the temperature gradient between the NIR-irradiated region and unexposed region. 66 As our catalase/Janus stomatocytes are also driven by a photothermal effect, they should also be susceptible to a swarming behavior. However, when the particles are simultaneously operated by light and chemical fuel, the "seesaw effect" could influence the swarming behavior of the catalase/Janus stomatocytes. Different motion states of the catalase/Janus stomatocytes might be obtained, according to the net outcome of the oppositely propelling forces. To explore these states, we used TP-CLSM to record the collective motion of the catalase/Janus stomatocytes under single and dual propulsion modes. As shown in Figures S15A to S15B, the catalase/Janus stomatocytes powered by both forces (laser on/with H 2 O 2 ) exhibited a halted state, due to the counterbalance between the two coexisting forces. As a result, the fluorescence intensity of the time lapsed CLSM images analysis by ImageJ did not change as there was no cluster formation in the "static state". In contrast, the dynamic state was achieved once switched to single mode, only applying the photothermal effect (laser on/no H 2 O 2 ). The catalase/Janus stomatocytes tended to form clusters during the observed periods, resulting in an increase in fluorescence intensity observed in the time lapsed CLSM images (Figures S15C to S15D).
In summary, we have designed a twin-engine supramolecular nanomotor based on bowl-shaped polymer vesicles or stomatocytes. Encapsulation of the enzyme catalase in the nanocavity of the stomatocytes provided the particles with the ability to undergo motion induced by the biocatalytic conversion of H 2 O 2 . By decorating the stomatocytes with a hemispherical gold shell opposite of the opening of the nanocavity, the particles were susceptible to NIR laser light and, consequently, were propelled via the photothermal effect. We demonstrated that the two modes of motion operate in different directions, which allowed us to create a "seesaw effect" with these supramolecular nanomotors; by tuning both the fuel concentration and incident laser power, particles were severely slowed down through balancing the two competitive propulsion modes. Moreover, the swarming behavior of the catalase/Janus stomatocytes could be tailored by adjusting the propulsion modes, which further demonstrated the versatility in motion control of these dual engine nanomotors, which could not be obtained by using only one propulsion mode. Given their attractive performance, the new twin-engine supramolecular nanomotors are expected to broaden the practical applications of nanomotors, ranging from on-demand assembly, environmental analysis, and sensing to activities in the biomedical field.   The manuscript was written through contributions of all authors.